1. Field of the Invention
The present invention relates to a serum separation sealant. More specifically, it relates to a serum separation sealant having an excellent balance of flow and specific gravity characteristics and an excellent storage stability.
2. Description of the Related Art
Recently, clinical analytical technologies detecting minute amounts of the intended biochemical substances have made remarkable advances. For this purpose, there have been demands for improved techniques of separating whole blood into the constituent components thereof, i.e., serum and blood cells. It is required in the above-mentioned analytical technologies that the separated serum portion should be free of any red blood cells which could affect the clinically measured data.
The present invention emplores the use of a thixotropic gelatinous material, dispensed in a test tube, in which blood (or blood plasma) is collected. Under the influence of a centrifugal force, the gelatinous material moves to a region between the upper serum (or plasma) and the lower solid blood clot portion due to the differences in the specific gravities, forming a partitioning barrier. The serum is then collected for clinical analysis. The formation of an effective barrier which separates the constituents and prevents the diffusion of blood cells back into the serum (or plasma) portion is the major function of a serum separator. Such a material must flow only under the influence of centrifugal force and maintain its dispensed position between the serum layer and the clot layer when the centrifugal force is relieved. For example, if the test tubes are tilted on their side, it is desirable for the separator material to maintain its position, whereby the blood cell portion does not flow and the partitioning barrier is not broken with the lapse of days. This is especially important during transport or storage where the tubes may be subjected to such positions. Excessive flowability would render the tubes useless.
The most suitable material for such a purpose possesses structural viscous properties, which is called thixotropic fluid properties, which impart a solid like behaviour during transient periods, and can break down during centrifugation, losing its solid properties, allowing for flow to occur. At the end of centrifugation, the gelatinous material has the ability to rebuild its network structure and retain its previous solid properties. Serum separators using such a principle were first suggested by U.S. Pat. No. 3,780,935 (Lukacs et al.) and U.S. Pat. No. 4,071,316 (Wright et al.), and consist of a network forming silica powder dispersed in a diorganosiloxane resin.
In these serum separatoes rheology and specific gravity were controlled according to polymer properties and silica contents. However, over extended storage periods, the silicone polymer had a tendency to react completely with the silica surface, resulting in reduced thixotropy. In other words, the mixture loses its solid like properties which results in a weakened barrier. To achieve appropriate thixotropy, high silica content was required. However, gels with excessive silica content tend to be hardened with time which was found to be detrimental to movement during centrifugation. Moreover, silicone polymers are expensive and difficult in adjusting properties of viscosity and specific gravity.
To alleviate the above-mentioned instability of the separation sealant of the prior art, Honda et al. (U.S. Pat. No. 4,534,798) have suggested the use of a polymer combination consisting of epoxy modified vegetable or animal oil and liquid chlorinated paraffins or liquid polyolefins. The epoxy modified vegetable/animal oil can form a strong association with silica powders and enhance the thixotropic properties. Liquid chlorinated paraffins or polyolefin constituents serve as a medium for dispersion. However, thixotropic properties were still rather low and required high polymer viscosities in order to attain barrier strength, making such gels difficult to work with during the production of gelatinous materials and the dispensing thereof into tubes.
In relation to silicone polymers, U.S. Pat. No. 4,083,784 (Zine, Jr.) implied the use of, as a third component, polysiloxane-polyoxyalkyl copolymer, which when added in sufficient amounts, competed with the main silicone polymer for reaction with the silica filler and maintained the required thixotropy over a long time. An added effect of this agent is to further enhance the thixotropic properties. Ichikawa et al. (U.S. Pat. No. 4,770,779) also entail using the same type of polysiloxane polymer, as a thixotropic agent, in a composite mixture of .alpha.-olefin-dimaleate copolymer and bentonite powder or silica. Another effect of using this thixotropic agent is the reduction in the concentration of network forming agents such as Aerosil and Bentonite powder.
Another example cited in U.S. Pat. No. 4,994,393 (Pradhan et al.) discloses the use of a dual resin system consisting of poly-.alpha.-pinene polymer and chlorinated hydrocarbons, a "network stabilizer" or thixotropic agent (e.g., glycerol, ethylene diamine, propylene glycol or ethylene glycol), and fumed silica or titanium dioxide. In this composition, the pinene resin per se had a low specific gravity and required addition of a chlorinated hydrocarbon resin to adjust to the desired level of the specific gravity. Consequently, when the polymer having a low specific gravity is mixed with immiscible resin for the adjustment of the specific gravity, gelatinous materials floating on the serum surface tend to be formed. This is especially prevalent when the material is stored over long periods of time and/or under severe conditions of 60.degree. C. or more, which can be encountered during transportation. In such cases, the mixture tends to be precipitated out of the dissolution conditions, due to immiscibility between silica powder and polymer. If there are components with a specific gravity less than the serum (s.g. =1.022-1.032), such as pinene and polysiloxane resin, they would have a strong tendency to break away from the main barrier body and float on the serum surface. Such a phenomena has no effect on immunoassay data, but has been proven to be a hindrance during analytical procedures.
JP-B-1-1-25026 discloses gelatinous materials. When a blood collecting tube is filled in advance with said gelatinous materials, it is preferable to use a material with a structural viscosity such that it will not flow in the distribution stage. On the other hand, when separating the blood, it is necessary to apply as small a centrifugal force (for example, less than 1500G) as possible in a short time (for example, less than 10 minutes) so as to prevent and to suppress effects of hemolysis on the clinical examination values. To promote movement of the gelatinous materials and form a barrier under a low centrifugal force, it is preferable that the viscosity of the gelatinous materials be low. The barrier of the gelatinous material, formed under centrifugation, must be sturdy or firm to prevent breakdown during the decantation of the serum. Thus, it is necessary that the gelatinous material possess transport stability and partitioning ability and also exhibits excellent movability.
As mentioned earlier, some gelatinous materials tend to "harden" with time, resulting in highly viscous materials, thus making movement difficult to achieve. To improve reliability, U.S. Pat. No. 4,189,382 (Zine, Jr.) suggests the insertion of a cylindrical like object, referred to as an "Energizer", inside the gel tube, after dispensing. Its main function is to apply extra force on the gel body, at the onset of centrifugation, and initiate movement. It can also serve as a carrier for clotting agents. Similar proposals have been made, using glass beads. U.S. Pat. No. 4,770,779 (Ichikawa et al.) suggests a specialized gel dispensing method such that a cone like cavity is formed on the upper surface. Such a condition, by virtue of the centrifugal force, also promotes the gel to readily flow. While these procedures are effective at maintaining reliability (or barrier formation), they require extra steps during dispensing, and add to the overall cost of the gel tube.
A practical requirement of the gelatinous materials is that they should exhibit little or no free flow properties, during transitory periods, regardless of the gel-containing tube position. It should maintain the initial dispensed position thereof, before actual use. If the gel exhibits excessive free flow, it could coat the entire tube surface, leading to remnant red blood cells adhering to the tube wall, on the serum side of the barrier. Furthermore, gel tubes, under this condition, tend to form weak or incomplete barriers. To minimize free flow, extra precaution must be taken to maintain the gel tubes in an upright position, for up to one day, upon completion of the dispensing procedure. This allows the networking to reform and retain its solid properties, broken down during the dispensing process. This again has shown to be a cumbersome procedure. Free flow can be inhibited by adjusting the gel formulation to achieve high "yield stresses", which is the minimum force per square unit area, required to make the gel flow. Higher yield stress promoted lower free flow properties. However, excessively high yield stress can be detrimental to the gel's ability to move, when centrifuged. In effect, provisions for reduced free flow formulations go contrary to the gel's functional ability to move during centrifugation.
Yield stress values have also been shown to affect the form and stability of the barrier. Before centrifugation, the collected gel tube-blood specimens must be maintained in a transitory condition, for the predetermined time period, to allow for clotting and contraction of the blood clot to take place and to prevent the formation of fibrinogen in the serum. After centrifugation, the blood clot is further compacted to the bottom of the tube. The blood clot does possess some elastic properties and would naturally tend to expand to its pre-centrifuged condition, thereby exerting an expansive force on the gel barrier which could effectively disturb the barrier form. Some gel systems overcome this problem simply by increasing polymer viscosity, which has also proven effective at maintaining barrier stability. The main drawback to this provision is that the resulting gel is very difficult to process and dispense.